1. Field of the Invention
The present invention relates to a process for producing S-hydroxynitrile lyase using a recombinant yeast cell into which S-hydroxynitrile lyase coding gene derived from cassava (Manihot esculenta) is introduced.
2. Description of the Prior Art
S-hydroxynitrile lyase derived from cassava (EC 4.1.2.37) is an enzyme useful for synthesis of optically active S-cyanohydrins from aromatic and/or aliphatic carbonyl compounds and hydrocyanic acid. Synthesis of optically active cyanohydrins using the enzyme is very useful for synthesis of various optically active intermediates.
However, it has been difficult to industrially utilize the present enzyme since cassava tissue contains an extremely low amount of the enzyme. One example of process for producing the enzyme is conventionally known, which comprises the steps of growing E. coli transformed with recombinant DNA into which S-hydroxynitrile lyase coding gene derived from cassava has been introduced. (Angew. Chem. Int. Ed. Engl. 35, 437-439, 1996; Biotechnol. Bioeng. 53, 332-338, 1997). However, such a process using E. coli as a host had several disadvantages including: a low productivity of the enzyme; and requirements to add expensive antibiotics and/or an inducer substrate to medium for producing a large amount of the enzyme. Because of these problems, it has been difficult to accomplish a process for efficient production of the enzyme with reduced cost.
The object of the present invention is to provide a process for producing a large amount of S-hydroxynitrile lyase by genetic-engineer technique.
After intensive studies, the present inventors finally succeeded in production of a large amount of S-hydroxynitrile lyase using yeast rather than using E. coli as a host for expression of S-hydroxynitrile lyase coding gene derived from cassava.
The present invention generally relates to a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, yeast cells transformed with recombinant DNA consisting of an expression vector into which S-hydroxynitrile lyase (EC 4. 1. 2. 37) coding gene derived from cassava (Manihot esculenta) has been incorporated, and collecting S-hydroxynitrile lyase from the culture.
Particularly, the present invention relates to the followings:
(1) a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, yeast cells transformed with recombinant DNA consisting of yeast episome expression vector into which S-hydroxynitrile lyase (EC 4. 1. 2. 37) coding gene derived from cassava (Manihot esculenta) has been incorporated, and collecting S-hydroxynitrile lyase from the culture;
(2) a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, the yeast Saccharomyces transformed with recombinant DNA consisting of yeast integrating expression vector into which S-hydroxynitrile lyase (EC 4. 1. 2. 37) coding gene derived from Cassava (Manihot esculenta) has been incorporated, and collecting S-hydroxynitrile lyase from the culture; and
(3) a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, the yeast Pichia transformed with recombinant DNA consisting of yeast integrating expression vector into which S-hydroxynitrile lyase (EC 4.1.2.37) coding gene derived from cassava (Manihot esculenta) has been incorporated, and collecting S-hydroxynitrile lyase from the culture.
This specification includes all or part of the contents as disclosed in the specification of Japanese Patent Applications Nos. 373246/1998, 373247/1998 and 373248/1998, which are priority documents of the present application.
The present invention will be hereinafter described in detail.
In accordance with the present invention, S-hydroxynitrile lyase coding gene of interest derived from cassava is cloned at first. The DNA sequence of the enzyme is well known and has been already disclosed. (Arch. Biochem, Biophys. 311, 496-502, 1994). Total mRNA including mRNA of the enzyme gene is extracted from cassava leaves and cDNA thereof is synthesized according to any conventional method. The enzyme coding gene is amplified by PCR using primers designed based on the well-known sequence data of S-hydroxynitrile lyase cDNA.
Next, an expression cassette was constructed by inserting a transcription promoter at upstream site and a transcription-terminator at downstream site of the enzyme gene to allow the enzyme gene obtained as described above to express in the recombinant yeast cells, and the constructed expression cassette is then introduced into an expression vector. Alternatively, where transcription promoter and terminator are already present in an expression vector into which the enzyme gene is to be introduced, the transcription promoter and terminator may be used and only the enzyme gene may be introduced therebetween, i.e., there is no need to construct an expression cassette. In either cases, multiple expression cassettes can be present in one expression vector.
Since the expression level of the enzyme greatly depends on the selection of a promoter to be used in an expression cassette, appropriate promoter should be selected. Examples of yeast promoter for efficient expression of the introduced gene in a yeast cell include native promoters such as PGK, GAP, TPI, GAL1, GAL10, ADH2, PHO5, CUP1 and MFxcex11, recombinant promoters such as PGK/xcex12 operator, GAP/GAL, PGK/GAL, GAP/ADH2, CYC/GRE and PGK/ARE, and mutated promoters such as Leu2-d. Particularly, GAP promoter is preferred.
Each of the promoters described above may have DNA consisting of the nucleotide sequence of a native promoter, or DNA consisting of the native promoter sequence having deletion, substitution and/or addition of one or more bases but still retaining the promoter activity. Deletion, substitution or addition of base(s) may be generated by using any conventional techniques such as site-directed mutagenesis.
On the other hand, transcription-terminator may be present downstream of the enzyme gene to allow efficient transcription-termination to obtain maximum gene expression. Examples of such transcription-terminator include ADH1, TDH1, TFF and TRP5.
Where the yeast Pichia is used as a host to be transformed, promoter in such an expression cassette as described above may be one which promotes enzyme expression within a methanol-utilizing strain in the yeast Pichia in the presence of methanol carbon source. Terminator in such an expression cassette may be one which allows efficient transcription-termination to obtain maximum gene expression. Particularly, AOX1 promoter and AOX1 terminator are preferred.
According to one embodiment of the present invention, yeast episome expression vector (autonomously replicating plasmid) is used as expression vector.
Yeast episome plasmid vector contains 2xcexc plasmid sequence, which is native to yeast. The vector can be replicated within a host yeast cell by utilizing the replication origin of the 2xcexc plasmid sequence.
Yeast episome expression vector to be used in the present invention may not be limited to particular vectors as long as it comprises at least ORI sequence of yeast 2xcexc plasmid sequence and can be autonomously replicated in a host yeast cell. Examples of such vector include YEp51, pYES2, YEp351 and YEp352 but are not limited thereto.
Preferably, the above-described yeast episome expression vector may be a shuttle vector which can replicate in a E. coli cell for subcloning in the recombinant E. coli. More preferably, such expression vectors may also contain a selective marker gene such as ampicillin-resistant gene. Also, such expression vectors contain a marker gene by which yeast clones can be selected depending on their auxotrophy and/or drug-resistance when recombinant yeast is prepared. Examples of marker gene include HIS3, TRP1, LEU2, URA3, LYS2, Tn903 kanr, Comr, Hygr, CUP1 and DHFR though they are not limited thereto. Preferably, a marker gene should be selected based on the genomic-type of the host Saccharomyces to be used for gene introduction.
Particular examples of the above-described yeast episome expression vector to be used in the present invention include: a vector constructed by incorporating GAP promoter into the multi-cloning site of yeast expression vector YEp352, S-hydroxynitrile lyase coding gene into downstream of the promoter and a terminator into further downstream (designated as YEp352-GC); a vector constructed by incorporating S-hydroxynitrile lyase coding gene into the multi-cloning site downstream of GAL 10 promoter in yeast expression vector YEp51 (designated as YEp51-C); a vector constructed by incorporating GAP promoter into the multi-cloning site of yeast expression vector YE351, S-hydroxynitrile lyase coding gene into downstream of the promoter and a terminator into further downstream (designated as YEp351-GC); and a vector constructed by incorporating S-hydroxynitrile lyase coding gene into the multi-cloning site downstream of GAL 1 promoter in yeast expression vector pYES2 (designated as pYES2-C).
According to another embodiment of the present invention yeast integrating expression vector (which can be integrated into chromosomal DNA) is used as expression vector.
Although yeast integrating plasmid vector has a DNA sequence (normally a selective marker gene sequence) homologous to that of yeast chromosome, it cannot be replicated as a plasmid in a yeast cell. Such yeast integrating plasmid vector can remain in yeast cells only when homologous replacement occurs between the sequence on the vector homologous to yeast chromosome and the yeast chromosome gene thereby integrating the plasmid vector into the chromosome. The integrated gene is known to be stably retained within the yeast cell even not under growth conditions where expression of selected marker gene is essential.
Yeast integrating expression vector to be used in the present invention is not limited to particular ones as long as it allows integration of S-hydroxynitrile lyase coding gene derived from cassava carried by the vector into yeast chromosome. For example, when incorporated into chromosome of Saccharomyces, yeast integrating vectors such as pRS303 and pRS304, or modified vectors constructed by excising yeast 2xcexc plasmid-derived sequence from vectors derived from yeast 2xcexc such as YEp51, pYES2, YEp351 and YEp352 and then cyclizing the vectors may be preferably used. Vectors for integration into the chromosome of a methanol-utilizing strain in the yeast Pichia are not limited to but include pPIC3.5K, pPIC9K and pAO815.
Yeast integrating expression vectors described above may be preferably shuttle vectors which can replicate in E. coli cells for subcloning in the recombinant E. coli cells. More preferably, such yeast expression vectors contain selective marker genes such as ampicillin-resistant genes. Alternatively, such expression vectors contain marker genes by which yeast clones can be selected depending on auxotrophy and drug resistance when recombinant yeast is prepared. Examples of marker gene for introduction into the yeast Saccharomyces include HIS3, TRP1, LEU2, URA3, LYS2, Tn903 kanr, Cmr, Hygr, CUP1 and DHFR though they are not limited thereto. A marker gene should be selected depending on the genomic-type of the host Saccharomyces strain into which the gene is to be introduced. Examples of marker gene for introduction into the yeast Pichia include HIS4 and kanr though they are not limited thereto. A marker gene should be selected depending on the genomic-type of the host Pichia strain into which the gene is to be introduced.
According to one embodiment of the present invention, the yeast Saccharomyces is used as a host though the host to be used in the present invention is not limited thereto as long as it can stably retain such an expression cassette after introduction of the cassette. Example of the yeast Saccharomyces include Saccharomyces cerevisiae KK4, Y334, Inv-Sc1 and W303 strains. Further, both haploid and diploid strains of these host yeast may be used.
According to another embodiment of the present invention, although a methanol-utilizing strain in the yeast Pichia is used as a host, yeast is not limited to particular ones as long as it can retain such an expression cassette after introduction of the cassette. Examples of methanol-utilizing strains in the yeast Pichia include Pichia pastoris KM71 and GS115 strains. Both haploid and diploid of these host yeast may be used.
In accordance with conventional transformation technique, recombinant yeast having an ability to produce S-hydroxynitrile lyase of interest can be obtained by introducing, into any of the above-described hosts, a yeast episome expression vector into which S-hydroxynitrile lyase coding gene derived from cassava is incorporated.
S-hydroxynitrile lyase can be produced by culturing the obtained recombinant yeast in a medium.
Medium may be conveniently supplemented with nitrogen sources such as yeast nitrogen base w/o amino acids (Difco Laboratories), essential amino acids and casamino acid, carbon sources such as glucose, galactose, raffinose and other saccharides, or alcohol such as glycerol and ethanol. The medium may be appropriately adjusted to pH 4-7.
According to the present invention, for culturing yeast transformed with yeast episome expression vector, composition of medium may be preferably altered depending on the selective marker gene on the vector to be used in order to prevent deletion of the enzyme gene present in the recombinant yeast cells. For example, medium which does not substantially contain uracil is selected for recombinant yeast transformed with yeast episome expression vector YEp352-GC where the selection marker gene is URA3. Alternatively, medium which does not substantially contain L-leucine is selected for recombinant yeast transformed with yeast episome expression vector YEp351-GC where the selective marker gene is LEU2.
Preferably, an inducer substrate may be added to the medium depending on the promoter to be used where enzyme production is required to be induced. For example, where promoter expression is promoted or inhibited by a particular carbon source, an appropriate carbon source should be selected for each case.
Where the expression of the enzyme gene is controlled by GAP promoter, which is one of the promoters preferable for the present invention, any carbon source which can be utilized by the host yeast cells may be used since the promoter will constitutively express.
On the other hand, for culturing yeast transformed with yeast integrating expression vector, medium may be suitably selected for growth of the host yeast to be used. There is no limitation to nutritional source nor need to add any antibiotics since the enzyme gene may be stably retained in the recombinant yeast cells. Preferably, medium may be adjusted to pH4-7.
Preferably, an inducer substrate may be added to the medium depending on the promoter to be used where enzyme production is required to be induced. For example, where promoter expression is promoted or inhibited by a particular carbon source, an appropriate carbon source should be selected for each case.
Where the expression of the enzyme gene is controlled by GAP promoter, which is one of the promoters suitable for the present invention, any carbon source which can be utilized by the host yeast cells may be used since the promoter will constitutively express.
On the other hand, where expression of the enzyme gene is controlled by AOX1 promoter, which is one of promoters suitable for the present invention, preferably glucose may not be added since it may repress/inhibit the expression though glycerol and raffinose do not affect the enzyme gene expression. Moreover, methanol may be preferably added to the culture for efficient expression of the enzyme gene and thus for production of a large amount of the enzyme since it will promote the gene expression.
The cells are cultured at 25-35xc2x0 C. for several hours to three days, for example, until the growth reaches at its stationary phase.
The recombinant yeast cells cultured as described above can produce a large amount of S-hydroxynitrile lyase.
Subsequently, S-hydroxynitrile lyase may be collected from the culture by using conventional enzyme collection methods including: cell lysis using cell-wall digesting enzyme (zymolyase); ultrasonication; disruption using glass beads; extraction with surfactants; self-digestion; and freezing-thawing method. Next, undissolved materials may be removed by, for example, filtration or centrifugation to give crude enzyme solution containing S-hydroxynitrile lyase.
S-hydroxynitrile lyase may be further purified from the crude enzyme solution by using any conventional protein purification method alone or in combination, including: ammonium sulfate fractionation; organic solvent precipitation; adsorption with ion exchanger; ion exchange chromatography; hydrophobic chromatography; gel filtration chromatography; affinity chromatography; and electrophoresis.
Effect of the Invention
According to the present invention, an efficient process for producing a large amount of S-hydroxynitrile lyase is provided using genetic-engineering technique.
Hereinafter the present invention will be described in detail by way of unlimiting examples.